A Raman spectroscopic microscope is very effective for observing a sample related to an organism. In the Raman spectroscopic microscope, an observed object is irradiated with a laser beam focused thereto, and a generated Raman scattering beam is detected. The Raman scattering beam is shifted from a wavelength of an excitation beam in a frequency thereof, and a spectrum is obtained by a spectroscope or the like. Positions of the observed object and an irradiation beam are changed relative to each other and the observed object is scanned by which spectroscopic spectra at respective positions can be obtained. An image can be formed based on the spectra. Raman spectra at respective observation positions reflect a vibrational excitement state of a molecule which is present at the positions, and are characteristic to the molecule. When a cell of an organism is observed by making use of the characteristic of the spectra, a distribution of organism molecules in a tissue is known.
FIG. 2 shows an energy level diagram showing a procedure of bringing about Raman scattering. Although there are stokes scattering and anti-stokes scattering in Raman scattering, only stokes scattering is shown in FIG. 2. Numeral 701 designates a vibrational ground state of a molecule, and numeral 702 designates a vibrational excitement state thereof. When a molecule is irradiated with a pumping beam having a frequency ωP, a beam having a frequency ωS is scattered by way of an intermediate state 703. At this occasion, the molecule results in one of vibrational excitement states. The frequency ωS of the scattering beam becomes a frequency of the stokes beam the frequency of which is lower than the frequency of the pumping beam. There are plural levels of vibrational excitement states of a molecule, and the vibrational excitement state differs depending on a kind of a molecule, a probability of transition from the intermediate state to the level of the vibrational excitement state differs, and therefore, a spectrum particular to the molecule is formed. A Raman shift frequency Ω is expressed by Ω=ωP−ωS, and becomes a positive value in a case of stokes scattering. In a, case of an anti-stokes beam, an initial state is a vibrational excitement state of a molecule, and a state of the molecule results in a vibrational ground state by way of an intermediate level. In this case, when notation ωAS designates a frequency of the anti-stokes beam, ωP<ωAS is established, that is, a frequency of an anti-stokes Raman scattering beam is higher than a frequency of a pumping beam.
The Raman scattering described above has a weak intensity of the scattering beam obtained, and therefore, time is taken for measurement. As a system of obtaining a strong scattering beam, there is a spectrometry which uses nonlinear Raman scattering referred to as CARS (Coherent Anti-Stokes Raman Scattering). Raman scattering can be obtained even by the method, and a vibrational state of a molecule can be known. A pulse laser having a high peak power is used for generating CARS. Thereby, a signal which is remarkably stronger than an a signal of Raman scattering is obtained, that is, a signal of a high signal to noise ratio is obtained, and a measurement time period can remarkably be shortened.
CARS is light emission by a third-order polarization, and a pumping beam, a stokes beam, and a probe beam are needed for generating CARS. Generally, a pumping beam substitutes for a probe beam in order to reduce a number of light sources. In this case, an induced third-order polarization is expressed as follows.Equation 1PAS(3)(ωAS)=|χr(3)(ωAS)+χnr(3)|EP2(ωP)E*S(ωS)  (1)Here, notation χr(3)(ωAS) designates a resonance term of a molecular vibration of an electric third-order susceptibility, and notation χnr(3) designates a non-resonance term thereof. Also, electric fields of the pumping beam and the probe beam are expressed as EP, and an electric field of the stokes beam is expressed as ES. The non-resonance term does not have frequency dependency. An asterisk attached to the shoulder of ES of Equation 1 indicates complex conjugate. An intensity of a CARS beam is expressed as follows.Equation 2ICARS(ωAS)∝|PAS(3)(ωAS)|2  (2)
An explanation will be given of a mechanism of generating the CARS beam in reference to an energy level diagram of a molecule (FIG. 3). The diagram shows a process of the resonance term. Similar to FIG. 2, a numeral 701 designates a vibrational ground state of a molecule and numeral 702 designates a vibrational excitement state thereof. A pumping beam having a frequency ωP and a stokes beam having a frequency ωS are simultaneously irradiated. At this occasion, the molecule is excited to a certain vibrational excitement level of 702 by way of an intermediate state 703. When the molecule which is brought into the excitement state is irradiated with a probe beam having a frequency ωP, the molecule returns to the vibrational ground state while generating a CARS beam having a frequency ωAS by way of an intermediate state 704. A frequency of the CARS beam at this occasion is expressed as ωAS=2·ωP−ωS.
FIG. 4 shows one process related to the non-resonance term of Equation 1. A frequency of the stokes beam configures a process not by way of the vibrational excitement state, but by way of an intermediate state 705. The intermediate state 705 related to an electron or the like is excited by simultaneously irradiating the pumping beam having the frequency ωP and a stokes beam having a frequency ω′S, and a non-resonant CARS beam having a frequency ωAS is generated by way of an intermediate state 704 by further irradiating a probe beam having a frequency ω′P. In a case where a laser beam having a narrow pulse width is used as the stokes beam, a beam having a broad frequency is irradiated, and therefore, there is a case of including the beam having the frequency ω′P or ω′S in FIG. 4. The resonant CARS beam and the non-resonant CARS beam are coherent to each other and interfered with each other.
Researches of spectra with regard to various kinds of molecules have been carried out since the Raman scattering was discovered in 1928, and accumulation of data is progressed. Therefore, it is preferable to identify a molecule in reference to the spectra data. CARS beam is expressed by Equations 1 and 2, and a portion in proportion to a Raman scattering spectrum is Im[χr(3)(ωAS)]. This is a complex part of the resonance term, which interferes with the non-resonance term χnr(3) as described above, and therefore, the Raman scattering spectrum cannot directly be obtained by the spectrum obtained by CARS.
A development of a method of extracting the Raman scattering spectrum from the CARS spectrum is an important problem, and various systems have been developed therefor (J. P. R. Day, K. F. Domke, G. Rago, H. Kano, H. Hamaguchi, E. M. Vartiainen, and M. Bonn “Quantitative Coherent Anti-stokes Scattering (CARS) Microscopy,” J. Phys. Chem. B, Vol. 115, 7713-7725 (2011)). For example, according to a maximum entropy method which is a method of recovering a phase spectrum from an intensity spectrum, a complex part of a resonance term is calculated by carrying out a mathematical calculation. Or, there is also a method making use of interference (C. L. Evans, E. O. Potma, X. S. and Xie, “Coherent Anti-Stokes Raman Scattering Spectral Interferometry: Determination of the Real and Imaginary Components of Nonlinear Susceptibility χ(3) for Vibrational Microscopy,” Opt. Lett. Vol. 29, 2923-2925 (2004)). According to the method, a CARS signal is generated by simultaneously focusing a pumping beam and a stokes beam to an observed sample by a condenser lens. On the other hand, a non-resonant CARS signal is obtained by irradiating a separate sample which generates the non-resonant CARS signal with a pumping beam and a stokes beam. The non-resonant CARS signal is made to be a local beam and the both CARS signals are interfered with each other. The local beam of the non-resonance signal is made to be a circularly polarized beam by a λ/4 plate, and a polarizing direction of the CARS beam from the observed sample is rotated by 45 degrees by a λ/2 plate. An interference beam of the beams is separated into two different linearly polarized beams, and the respective beams are subjected to spectrometry by a spectroscope. When an electric field of CARS from the observed sample is designated by notation EAS (ω) and an electric field of the local beam is designated by notation ELO, respective interference signals are expressed as follows.Equation 3SC(ω)=|ELO|2+EAS(ω)|2+2|ELOEAS(ω)|cos Φ(ω)  (3)Equation 4SS(ω)=|ELO|2+|EAS(ω)|2+2|ELOEAS(ω)|sin Φ(ω)  (4)Here, notation Φ(ω) expresses a phase difference between the local beam and the CARS signal beam, and is expressed as Φ(ω)=ωτ+θS(ω)+θinst(ω). Notation COT designates an optical path difference between the two beams, notation θS(ω) designates a phase difference by a resonant beam, and notation θinst(ω) designates a phase difference originated from an apparatus. |ELO|2 and |EAS(ω)|2 in Equations 3 and 4 can be calculated by cutting off one of them. Therefore, tan Φ(ω) can be calculated and also Φ(ω) can be determined from Equations 3 and 4. First, ωτ+θinst(ω) is determined by measuring a sample which generates only the non-resonant CARS beam as an observed sample. Next, an observed sample which generates the resonant CARS is measured. θS(ω) can be determined thereby, and therefore, the complex part of the resonance component can be calculated as |EAS(ω)|sin θS(ω). Thereby, what corresponds to the Raman scattering spectrum can be obtained.
The above-described method is a method of determining ωτ+θinst(ω) accurately by using the sample which generates the non-resonant CARS beam. However, when there are known a peak frequency of the CARS spectrum which is obtained previously and a frequency by which the spectrum becomes flat in a case of small frequency dependency of θinst(ω), there may be set θinst(ω) as an initial value for realizing the spectrum. In this case, a reference sample which generates the non-resonance CARS beam is not needed.
There is a Raman scattering spectrum region (1800 through 800 cm−1) which is referred to as a fingerprint region as a spectrum region which is sensitive to a molecular structure. It is preferable to obtain a spectrum of a similar region also in detecting a CARS beam. According to the system introduced in J. P. R. Day, K. F. Domke, G. Rago, H. Kano, H. Hamaguchi, E. M. Vartiainen, and M. Bonn “Quantitative Coherent Anti-stokes Scattering (CARS) Microscopy,” J. Phys. Chem. B, Vol. 115, 7713-7725 (2011), a spectrum width of the stokes beam for excitement is about 140 cm−1, and the system cannot cover the region. There is introduced a system of using a photonic fiber as a light source for compensating for the drawback in M. Okuno, H. Kano, P. Leproux, V. Couderc, J. P. R. Day, M. Bonn, and H. Hamaguchi, “Quantitative CARS Molecular Fingerprinting of Single Living Cells with the Use of the Maximum Entropy Method,” Angew. Chem. Int. Ed. Vol. 49.6773-6777 (2010). There is generated a broadband beam which is referred to as Super Continuum Beam (SC beam) by irradiating a photonic fiber with an extremely short pulse laser.